Lis1, the Drosophila homolog of a human lissencephaly disease gene, is required for germline cell division and oocyte differentiation.

Lissencephaly is a severe congenital brain malformation resulting from incomplete neuronal migration. One causal gene, LIS1, is homologous to nudF, a gene required for nuclear migration in A. nidulans. We have characterized the Drosophila homolog of LIS1 (Lis1) and show that Lis1 is essential for fly development. Analysis of ovarian Lis1 mutant clones demonstrates that Lis1 is required in the germline for synchronized germline cell division, fusome integrity and oocyte differentiation. Abnormal packaging of the cysts was observed in Lis1 mutant clones. Our results indicate that LIS1 is important for cell division and differentiation and the function of the membrane cytoskeleton. They support the notion that LIS1 functions with the dynein complex to regulate nuclear migration or cell migration.     

Mice deficient for spermatid perinuclear RNA-binding protein show neurologic, spermatogenic, and sperm morphological abnormalities

Spermatid perinuclear RNA-binding protein (SPNR) is a microtubule-associated RNA-binding protein that localizes to the manchette in developing spermatids. The Spnr mRNA is expressed at high levels in testis, ovary, and brain and is present in these tissues in multiple forms. We have generated a gene trap allele of the murine Spnr, named Spnr(+/GT). Spnr(GT/GT) mutants show a high rate of mortality, reduced weight, and an abnormal clutching reflex. In addition to minor anatomical abnormalities in the brain, males exhibit defects in spermatogenesis that include a thin seminiferous epithelium and disorganization of spermatogenesis. Most of the sperm from mutant males display defects in the flagellum and consequently show decreased motility and transport within the oviducts. Furthermore, sperm from mutant males achieve in vitro fertilization less frequently. Our findings suggest that SPNR plays an important role in normal spermatogenesis and sperm function. Thus, the Spnr(GT/GT) mutant male mouse provides a unique model for some human male infertility cases.     

Lissencephaly associated mutations suggest a requirement for the PAFAH1B heterotrimeric complex in brain development

Human brain malformations, such as Miller-Dieker syndrome (MDS) or isolated lissencephaly sequence (ILS) may result from abnormal neuronal migration during brain development. MDS and ILS patients have a hemizygous deletion or mutation in the LIS1 gene (PAFAH1B1), therefore, the LIS1 encoded protein (Lis1) may play a role in neuronal migration. Lis1 is a subunit of a brain platelet-activating factor acetylhydrolase (PAFAH1B) where it forms a heterotrimeric complex with two hydrolase subunits, referred to as 29 kDa (PAFAH1B3) and 30 kDa (PAFAH1B2). In order to determine whether this heterotrimer is required for the developmental functions of PAFAH1B, we examined the binding properties of 29 and 30 kDa subunits to mutant Lis1 proteins. The results defined the critical regions of Lis1 for PAFAH1B complex formation and demonstrated that all human LIS1 mutations examined resulted in abolished or reduced capacity of Lis1 to interact with the 29 and 30 kDa subunits, suggesting that the PAFAH1B complex participates in the process of neuronal migration.     

Global developmental gene expression and pathway analysis of normal brain development and mouse models of human neuronal migration defects

Heterozygous LIS1 mutations are the most common cause of human lissencephaly, a human neuronal migration defect, and DCX mutations are the most common cause of X-linked lissencephaly. LIS1 is part of a protein complex including NDEL1 and 14-3-3ε that regulates dynein motor function and microtubule dynamics, while DCX stabilizes microtubules and cooperates with LIS1 during neuronal migration and neurogenesis. Targeted gene mutations of Lis1, Dcx, Ywhae (coding for 14-3-3ε), and Ndel1 lead to neuronal migration defects in mouse and provide models of human lissencephaly, as well as aid the study of related neuro-developmental diseases. Here we investigated the developing brain of these four mutants and wild-type mice using expression microarrays, bioinformatic analyses, and in vivo/in vitro experiments to address whether mutations in different members of the LIS1 neuronal migration complex lead to similar and/or distinct global gene expression alterations. Consistent with the overall successful development of the mutant brains, unsupervised clustering and co-expression analysis suggested that cell cycle and synaptogenesis genes are similarly expressed and co-regulated in WT and mutant brains in a time-dependent fashion. By contrast, focused co-expression analysis in the Lis1 and Ndel1 mutants uncovered substantial differences in the correlation among pathways. Differential expression analysis revealed that cell cycle, cell adhesion, and cytoskeleton organization pathways are commonly altered in all mutants, while synaptogenesis, cell morphology, and inflammation/immune response are specifically altered in one or more mutants. We found several commonly dysregulated genes located within pathogenic deletion/duplication regions, which represent novel candidates of human mental retardation and neurocognitive disabilities. Our analysis suggests that gene expression and pathway analysis in mouse models of a similar disorder or within a common pathway can be used to define novel candidates for related human diseases.     

Complete loss of Ndel1 results in neuronal migration defects and early embryonic lethality

Regulation of cytoplasmic dynein and microtubule dynamics is crucial for both mitotic cell division and neuronal migration. NDEL1 was identified as a protein interacting with LIS1, the protein product of a gene mutated in the lissencephaly. To elucidate NDEL1 function in vivo, we generated null and hypomorphic alleles of Ndel1 in mice by targeted gene disruption. Ndel1(-/-) mice were embryonic lethal at the peri-implantation stage like null mutants of Lis1 and cytoplasmic dynein heavy chain. In addition, Ndel1(-/-) blastocysts failed to grow in culture and exhibited a cell proliferation defect in inner cell mass. Although Ndel1(+/-) mice displayed no obvious phenotypes, further reduction of NDEL1 by making null/hypomorph compound heterozygotes (Ndel1(cko/-)) resulted in histological defects consistent with mild neuronal migration defects. Double Lis1(cko/+)-Ndel1(+/-) mice or Lis1(+/-)-Ndel1(+/-) mice displayed more severe neuronal migration defects than Lis1(cko/+)-Ndel1(+/)(+) mice or Lis1(+/-)-Ndel1(+/+) mice, respectively. We demonstrated distinct abnormalities in microtubule organization and similar defects in the distribution of beta-COP-positive vesicles (to assess dynein function) between Ndel1 or Lis1-null MEFs, as well as similar neuronal migration defects in Ndel1- or Lis1-null granule cells. Rescue of these defects in mouse embryonic fibroblasts and granule cells by overexpressing LIS1, NDEL1, or NDE1 suggest that NDEL1, LIS1, and NDE1 act in a common pathway to regulate dynein but each has distinct roles in the regulation of microtubule organization and neuronal migration.     

Identification of a heat-shock protein Hsp40, DjB1, as an acrosome- and a tail-associated component in rodent spermatozoa

Iba1 is a 17-kDa EF-hand protein highly expressed in the cytoplasm of elongating spermatids in testis. Using Iba1 as a bait, we performed yeast Two-hybrid screening and isolated a heat-shock protein Hsp40, DjB1, from cDNA library of mouse testis. To characterize DjB1 that is encoded by Dnajb1 gene, we carried out immunoblot analyses, in situ hybridization, and immunohistochemistry. Immunoblot analyses showed that DjB1was constitutively expressed in mouse testis and that its expression level was not changed by heat shock. Dnajb1 mRNA was exclusively expressed in spermatocytes and round spermatids in mouse testis, and Dnajb1 protein DjB1 was predominantly expressed in the cytoplasm of spermatocytes, round spermatids, and elongating spermatids. In mature mouse spermatozoa, DjB1 was localized in the middle and the end pieces of flagella as well as in association with the head (acrosomal region). Association of DjB1 with the acrosomal region in sperm head was also observed in rat spermatozoa. These data suggested that DjB1, which was constitutively expressed in postmeiotic spermatogenic cells in testis, was integrated into spermatozoa as at least two components, that is, sperm head and tail of rodent spermatozoa.     

Genetic mosaic dissection of Lis1 and Ndel1 in neuronal migration

Coordinated migration of newly born neurons to their prospective target laminae is a prerequisite for neural circuit assembly in the developing brain. The evolutionarily conserved LIS1/NDEL1 complex is essential for neuronal migration in the mammalian cerebral cortex. The cytoplasmic nature of LIS1 and NDEL1 proteins suggest that they regulate neuronal migration cell autonomously. Here, we extend mosaic analysis with double markers (MADM) to mouse chromosome 11 where Lis1, Ndel1, and 14-3-3? (encoding a LIS1/NDEL1 signaling partner) are located. Analyses of sparse and uniquely labeled mutant cells in mosaic animals reveal distinct cell-autonomous functions for these three genes. Lis1 regulates neuronal migration efficiency in a dose-dependent manner, while Ndel1 is essential for a specific, previously uncharacterized, late step of neuronal migration: entry into the target lamina. Comparisons with previous genetic perturbations of Lis1 and Ndel1 also suggest a surprising degree of cell-nonautonomous function for these proteins in regulating neuronal migration.     

Ca(2+)/calmodulin-dependent protein kinase IV is expressed in spermatids and targeted to chromatin and the nuclear matrix

Ca(2+)/calmodulin-dependent protein kinase IV and calspermin are two proteins encoded by the Camk4 gene. Both are highly expressed in the testis, where in situ hybridization studies in rat testes have demonstrated that CaMKIV mRNA is localized to pachytene spermatocytes, while calspermin mRNA is restricted to spermatids. We have examined the expression patterns of both CaMKIV and calspermin in mouse testis and unexpectedly find that CaMKIV is expressed in spermatogonia and spermatids but excluded from spermatocytes, while calspermin is found only in spermatids. CaMKIV and calspermin expression in the testis are stage-dependent and appear to be coordinately regulated. In germ cells, we find that CaMKIV is associated with the chromatin. We further demonstrate that a fraction of CaMKIV in spermatids is hyperphosphorylated and specifically localized to the nuclear matrix. These novel findings may implicate CaMKIV in chromatin remodeling during nuclear condensation of spermatids.     

Complementary DNA cloning of rat spetex-1, a spermatid-expressing gene-1, encoding a 63 kDa cytoplasmic protein of elongate spermatids

We used differential display in combination with complementary DNA (cDNA) cloning approach to isolate a novel rat gene designated as spetex-1, which had an open reading frame of 1,668-length nucleotides encoding a protein of 556 amino acids. Spetex-1 mRNA was highly expressed in testis, and weekly expressed in lung, intestine, and spleen. Spetex-1 expression in the rat testes was detected first at 3 weeks in postnatal development and continued to be detected up to adulthood. A search in the databases showed that the amino acid sequence of spetex-1 was 82% identical to that of its mouse homologue found in the databases. Both rat spetex-1 and the mouse homologue contained Ser-X (X = His, Arg, or Asn) repeats in the middle portion of the proteins. In situ hybridization revealed that spetex-1 mRNA was expressed in haploid spermatids of step 7-18 within the seminiferous epithelium. Immunohistochemical analysis with confocal laser-scanning microscopy demonstrated that spetex-1 protein was not expressed in spermatogonia, spermatocytes, and round spermatids in adult rat testis, but was specifically detected in the residual cytoplasm of elongate spermatids of step 15-18 as well as in residual bodies engulfed by Sertoli cells. We interpreted these data as a potential role of spetex-1 in spermatogenesis, especially in cell differentiation from late elongate spermatids to mature spermatozoa.     

The structure of the N-terminal domain of the product of the lissencephaly gene Lis1 and its functional implications

Mutations in the Lis1 gene result in lissencephaly (smooth brain), a debilitating developmental syndrome caused by the impaired ability of postmitotic neurons to migrate to their correct destination in the cerebral cortex. Sequence similarities suggest that the LIS1 protein contains a C-terminal seven-blade beta-propeller domain, while the structure of the N-terminal fragment includes the LisH (Lis-homology) motif, a pattern found in over 100 eukaryotic proteins with a hitherto unknown function. We present the 1.75 A resolution crystal structure of the N-terminal domain of mouse LIS1, and we show that the LisH motif is a novel, thermodynamically very stable dimerization domain. The structure explains the molecular basis of a low severity form of lissencephaly.     

Molecular cloning of rat Spetex2 family genes mapped on chromosome 15p16, encoding a 23-kilodalton protein associated with the plasma membranes of haploid spermatids

We used differential display in combination with cDNA cloning to isolate a novel rat gene, designated as Spetex2, that has an open reading frame of 582 nucleotides, encoding a protein of 194 amino acids. Spetex2 mRNA was highly expressed in testis and spleen, and its expression in rat testis was developmentally up-regulated. In situ hybridization revealed that Spetex2 mRNA was predominantly expressed in haploid spermatids at steps 1-13 within the seminiferous epithelium. A BLAST search against rat genome databases at the National Center for Biotechnology Information revealed that the Spetex2 gene is composed of four exons and is mapped to at least 18 loci in a cluster on rat chromosome 15p16, indicating that the genes occur as a repeated tandem array over a long stretch of genomic DNA. By immunocytochemical analysis with confocal laser-scanning microscopy, SPETEX2 protein was detected as a dot-like distribution on the cell periphery of haploid spermatids (steps 1-13) but was not observed in other spermatogenic cells. On the basis of these data, we hypothesize that SPETEX2 might be correlated with cell differentiation of spermaytids in rat testis.